| Background and ObjectiveAcute lung injury(ALI)is a diffuse,inflammatory lung parenchymal injury disease involving a variety of inflammatory mediators and effector cells,leading to cascaded amplification of the cascade of inflammatory responses.Multiple local-infiltrated inflammatory cells such as neutrophils and mononuclear macrophages are involved in the pathophysiology of ALI.During its progression,the alveolar macrophages(AM)exhibit dual roles in both the initiation and resolution of inflammation through various mechanisms.Infection is the leading cause of acute lung injury in children,and bacterial and viral infections rank first.The pathogenesis of bacterial acute lung injury in children,especially sepsis-induced acute lung injury,has been studied in clinical and animal experiments.However,the pathogenesis of acute lung injury caused by viral infection is seldom studied,especially the mechanisms or signaling pathways of alveolar macrophages in acute lung injury after infection.Neutral granulocytes accumulate in microvessels,adhere to endothelial cells and then release various inflammatory mediators and toxic molecules,which can lead to alveolar capillary membranes damage,permeable pulmonary edema and then ALI,however,inflammatory factors secreted by stimulated AM can induce the process.CCL3 promotes the pulmonary inflammation by stimulating monocytes and alveolar macrophages.Specially binding of CCL3 to corresponding receptors can lead to water-fall inflammatory cell activation.The receptors of CCL3 include CCR1,CCR5 and CCR5,which belong to G-protein coupled proteins.Soehnlein found that CCR5 played important role in the recruitment of mononuclear macrophages.Epigenetic modification is involved in the differentiation and polarization of macrophages by transcriptional regulation,which determines the migration activity and function of different macrophage subpopulations.JMJD superfamilies(JHDMs)belong to histone demethylases,including JMJD1 A,JMJD1C,JMJD2A,JMJD3,etc.,which can catalyze the histones demethylation,thereby regulating gene transcription,chromatin structure,damage repair and epigenetic memory.Studies have shown that the expression of histone demethylase JMJD3 is up-regulated in LPS-acting macrophages,which can regulate target gene transcription by affecting H3 lysine 27 trimethylation(H3K27me3)levels.However,epigenetic modification of macrophages by virus-related products remains unclear.The recruitment of AM plays an important role in the pathogenesis of ALI,and viral infection can greatly promote the accumulation of AM in the lungs.Our study aims to explore the role and intrinsic mechanism of macrophage activation in ALI.The first part of the study analyzed the effect of viral product poly I:C on the chemokines expression and chemotactic activity of THP-1 derived macrophage,and explored the role and regulation mechanism of histone demethylase(JHDMs)in this process,thus analyzing the mechanism of macrophage activation and recruitment involved in the viral ALI.The second part of the study established an animal model of viral ALI,then detected the expression of corresponding chemokines and receptors,thereby verifying the mechanism of alveolar macrophage activation in viral ALI in vivo.Methods and Results1.Section 1(experiment in vitro):The study of macrophages chemotaxis,CCR5 expression and its TLR3/JMJD1A signaling by poly I:C.The main experimental technique of this study:Cell culture and treatment,Flow cytometry,RNA extraction and reverse transcription,Real-time PCR,siRNA transfection,Macrophage migration experiment,Chromosome immune coprecipitation(ChIP),Western blot,et al.(1)The expression profile of chemokine receptors in polyI:C-stimulated THP-1-derived macrophages:Chemokines and their receptors play important roles in macrophages infiltration during the development of inflammatory diseases,including ALI.We first detected the expression of several chemokine receptors in THP-1 monocytes and THP-1-derived macrophages by real-time PCR,and the results were displayed:CCR1,CCR4,CCR5 and CCR6 are expressed in both THP-1 monocytes and macrophages under static condition,and CCR1 expression was significantly higher in macrophages compared with THP-1 monocytes.We went on to analyze the expression of CCR1,CCR4,CCR5 and CCR6 in polyI:C-stimulated THP-1 monocytes and macrophages.The expression of CCR5 is significantly elevated after polyI:C treatment in THP-1-derived macrophages,but no significant change in CCR5 expression was observed in polyI:C-stimulated monocytes.The mRNA expression of CCR1,CCR4 and CCR6 was not significantly changed after polyI:C treatment in either THP-1 monocytes or macrophages.The remarkable increase in CCR5 expression in polyI:C stimulated THP-1-derived macrophages has been confirmed by flow cytometric(45.9%of polyI:C vs 20.8%of control).Macrophages recognized polyI:C stimulation through TLR3 signaling.Therefore,we went on to detect the changes of CCR5 expression in THP-1 macrophages after TLR3 silencing.No obvious changes were observed between control siRNA and TLR3 siRNA groups under static condition.However,the expression of CCR5 was significantly decreased in TLR3-knockdown THP-1-derived macrophages after polyl:C stimulation.(2)Polyl:C promoted the chemotaxis of THP-1-derived macrophages to CCL3,mediated by TLR3 signaling:CCR5 can be activated by several ligands including CCL3.We presumed that polyl:C stimulation induced THP-1-derived macrophages migration via CCL3/CCR5 axis.To prove this,we established a transwell assay by adding the polyI:C-or medium-treated THP-1-derived macrophages to the upper wells and recombinant human CCL3(rhCCL3)to the lower wells.THP-1-derived macrophages migrated to CCL3 under static condition,whereas polyI:C stimulation further increased the chemotaxis activity toward rhCCL3.In addition,after silencing TLR3 expression in polyI:C-stimulated THP-1-derived macrophages,the migration toward rhCCL3 was significantly decreased.(3)Analysis of JHDM family member expression in polyl:C-stimulated macrophages:Previous studies showed that histone methylation was involved in inflammatory reactions of macrophages.We went on to identify the expression of 23 members from Jumonji C domain-containing histone demethylase(JHDM)family in medium or polyI:C-stimulated THP-1-derived macrophages by real-time PCR.In the top 10 highly-expressed JHDM family members,JMJD1A,JMJD1C,JMJD2A,JARID1A and HSPBAP1 were significantly up-regulated after polyI:C stimulation,with JARID1A showing the greatest change(3.65±0.45 fold).From the results above,we noticed that two JHDM2 subgroup members,JMJD1A and JMJD1C,exhibited high abundance and significantly over-expression in polyI:C-stimulated THP-1-derived macrophages.Knockdown of TLR3 significantly reversed the enhancing effect of polyI:C on both their expression in THP-1-derived macrophages.Therefore,these two JHDM family members were chosen as the targets for the following experiments.(4)PolyI:C-regulated JMJD1A increased the expression of CCR5 and possibly through its substrate,H3K9me2:To investigate whether increased JMJD1A and JMJD1C were involved in CCR5 expression,these two genes were silenced in medium-or polyI:C-stimulated THP-1-derived macrophages.While JMJD1A knockdown induced a significant decrease of CCR5 mRNA expression in both control and polyI:C-treated macrophages,JMJD1C knockdown only caused a slight decrease in both groups.The down-regulation of CCR5 expression by JMJD1A silencing was also confirmed by flow cytometry.H3K9me2 was known as the substrate of JMJD1A.Therefore,we went on to testify whether the modification of JMJD1A on CCR5 expression was dependent on the regulation of H3K9 methylation.PolyI:C stimulation induced a great decrease for H3K9me2,while the level of H3K9me3 was constant.The absence of JMJD1A in medium-treated macrophages caused over-expression of H3K9me2,however,its knockdown in polyI:C-treated macrophages led no significant change.The methylation of histone H3K9 in upstream promoter regions usually led to reduced gene expression.Since its methylation was reduced on polyI:C stimulation,we performed ChIP-qRT-PCR experiments and analyzed histone H3K27 methylation in upstream promoter region of CCR5 with or without polyI:C stimulation.Our results found that H3K9me2 was significantly reduced at promoter region under polyI:C treatment.2.Section 2(animal experiment):The study of viral acute lung injury in mouse model to verify macrophages activity and its mechanism.Forty 4-week-old SPF male mice weighing 13-15g were randomly divided into 4 groups(T1 and T2 in experimental group,Cnl and Cn2 in control group).PolyI:C was used to inhale into trachea through oropharynx in T1 and T2 groups,and Cnl and Cn2 were given the same amount of saline.The body weight of mice in group T1 and Cnl was measured at regular intervals,and the survival state of mice was observed and recorded for 15 days.The expression of CCL3,TNF-a,IL-1? and IL-6 in the supernatant of bronchoalveolar lavage fluid was detected by ELISA.The precipitated cells were cultured in culture medium;alveolar macrophages were collected and purified;and real-time quantitative PCR method was used to detect the levels of CCR5,TLR3 and JMJD1A in alveolar macrophages.Lung tissue was taken to measure lung index(lung index = lung wet weight/body weight X 100%),lung wet dry weight ratio(W/D = lung wet weight/lung dry weight),and lung tissue sections were stained with HE to observe histopathological changes and pathological injury score.(1)T1 and T2 mice treated with poly I:C gradually appeared to be depressed in spirit,decreased in activity,decreased in foraging,faster respiratory rate,loose fur and so on,and their weight showed a downward trend.Mice died after sixth days of polyI:C treatment,and 7 mice died within 15 days.Anatomically,pulmonary edema,congestion and bleeding,intra-bronchial hemorrhagic foam like fluid can be seen.There was no abnormality in saline control group.(2)On the 6th day after poly I:C treatment,the PaO2 and SaO2 of experimental group T2 were significantly lower than those of control group Cn2(P<0.01),and the PaCO2 of experimental group T2 was significantly higher than that of control group C n2(P<0.05).The PH value of experimental group was not significantly different from that of control group(P>0.05).On the 6th day after polyl:C treatment,the average lung index of Cn2 group(0.66±0.02),T2 group(1.98±0.06)and T2 group were significantly higher than Cn2 group(P<0.01).The W/D value of T2 in experimental group was higher than that in group Cn2(P<0.01).(3)HE staining pathological damage score of lung tissue slices:T2 group mice alveolar wall edema,thickening,alveolar cavity smaller,interstitial lung lesions,loose edema;pulmonary tissue hemorrhage,alveolar cavity and interstitial large number of red blood cells.There was no obvious abnormality in lung tissue of mice in group Cn2.The pathological injury score of lung tissue in T2 group(5.76±0.51)was significantly higher than that in control group(1.47±0.23),and the difference was statistically significant(P<0.01).(4)The expressions of CCL3,TNF-a,IL-1? and IL-6 in alveolar lavage fluid of T2 group were significantly higher than those of Cn2 group(P<0.01).The expressions of CCR5,TLR3 and JMJD1A in alveolar macrophages of group T2 were significantly higher than those in Cn2 group(P<0.01).Conclusion1.polyI:C promotes the expression of CCR5 in macrophages through TLR3,thereby promoting its chemotactic activity towards CCL3.This process may be achieved by up-regulating the expression of JMJD1A,which inhibits the binding of H3K9me2 to the CCR5 promoter region,thereby enhancing the transcriptional activity of CCR5-encoded genes.2.Successful modeling of viral acute lung injury in mice after polyI:C treatment suggests that acute lung injury caused by virus infection is related to inflammatory injury and macrophage activation.It is confirmed that alveolar macrophage recruitment is induced by TLR3/JMJD1A signaling pathway to promote CCR5 expression and chemotactic activity of AM. |
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